Use of pentamethylene quinoxaline di-oxides as antibacterial agents and animal growth promotants

ABSTRACT

A SERIES OF 7,8,9,10-TETRAHYDRO-6H-CYCLOHEPTA(B)QUINOXALINE-5,11-DIOXIDES AND NON-TOXIC SALTS THEREOF USEFUL AS ANTIBACTERIAL AGENTS AND AS ANIMAL GROWTH PROMOTANTS.

United States Patent O USE OF PENTAMETHYLENE QUINOXALINE DI- OXIDES ASANTIBACTERIAL AGENTS AND ANI- MAL GROWTHPROMOTANTS James David Johnston,Old Saybrook, Conn., assignor to Chas. Pfizer & Co., Inc., New York,N.Y., a corporation of Delaware No Drawing. Application Oct. 18, 1966,Ser. No. 587,422, now Patent No. 3,471,492, dated Oct. 7, 1969, which isa continuation-in-part of abandoned application Ser. No. 463,932, June14, 1965. Divided and this application Jan. 6, 1969, Ser. No. 803,514

Int. Cl. A61k 27/00 US. Cl. 424250 6 Claims ABSTRACT OF THE DISCLOSURE Aseries of 7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxal1ne-5,11-dioxidesand non-toxic salts thereof useful as antibacterial agents and as animalgrowth promotants.

CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisionof application Ser. No. 587,422 as filed Oct. 18, 1966 (now US. Patent3,471,- 492) which, in turn, is a continuation-in-part of applicationSer. No. 463,932 filed June 14, 1965 and now abandoned.

This invention relates to novel pentamethylene quinoxaline-1,4-dioxidesuseful as therapeutic agents and as growth promotants and to processesfor the treatment of antibacterial infections and for the promotion ofgrowth in animals. More particularly, it relates to a series of 7,8,9,10tetrahydro 6H cyclohepta[b]quinoxaline- 5,11-dioxides useful for thetreatment of gram-negative infections in animals, and as growthpromotants.

The novel compounds of this invention have the general formulae:

IV V

wherein R is selected from the group consisting of hydroxy, loweralkoxy, mercapto, lower alkylmercapto, lower alkanoyloxy, cyano,carboxy, carbo(lower)alkoxy, chloro and bromo; R is selected from thegroup consisting of hydrogen and R Of the lower alkoxy,carbo(lwer)alkoxy, lower alkylmercapto and lower alkanoyloxy groups,those having from one to four carbon atoms in the alkoxy,alkylmeradministered. The preferred acid addition salts of the abovementioned bases which may be employed are the hydrochloride,hydrobromide, phosphate, nitrate and sulfate. The non-toxic metal saltsof particular interest are the sodium, potassium, calcium and magnesiumsalts.

The disubstituted 7,8,9,10 tetrahydro 6H cyclohepta[b]quinoxaline-5,1l-dioxides of Formula I and their free bases exist in two epimericforms. Both epimers of a given compound are active antibacterial agents.

The antibacterial activity of several 2,3-polymethylene quinoxaline1,4-dioxides has been disclosed in the literature. Ursprung, US. Patent2,891,062, describes the activity of certain l-hydroxyand1,4-dihydroxy-l,2,3,4- tetrahydrophenazine-S,lO-dioxides asanti-infective agents. especially against Proteus vulgaris. Theantifungal activity of the epimeric1,4-dibromo-l,2,3,4-tetrahydrophenazine- 5,10 dioxides is disclosed byGordon, et al. in US. Patent 2,921,937. Iland (Nature 161, 1010, 1948)and McIlWain (J. Chem. Soc. 323-325, 1943) describe the antibacterialproperties of l,2,3,4-tetrahydrophenazine-5,10-dioxide.

Hurst, et al., Brit. J. Pharmacol. 8, 297-305 (1953) report on theactivity of several 1,2,3,4-tetrahydrophenazine-5,10-dioxides and of2,3-pentamethylenequinoxaline- 1,4-dioxides against the largest virusesof the psittacosislymphogranuloma group and note that toxicside-reactions preclude their use in man. The latter compound was foundto be, at most, only slightly active against the viruses studied.Landquist, J. Chem. Soc. 2551-2553 (1956) describes the preparation of aseries of 2,3-polymethylene quinoxaline-1,4-dioxides wherein thepolymethylene moiety is tri-, tetraand penta-methylene as potentialchemotherapeutic agents. The preparation and bacteriostatic study ofseveral substituted 1,2,3,4-tetrahydrophenazine- 5,10-dioxidesv arereported by King, et al., J. Chem. Soc. 3012-3016 (1949).

The valuable compounds of this invention are readily prepared by methodswell known in the art. The most convenient methods from the standpointof availability of materials, ease and simplicity of reaction, yield andpurity of product for preparing compounds of Formula I are thecondensation of 1,2-cycloheptanedione with o-phenylenediamine, and ofthe reaction of cycloheptanone with o-aminoazobenzene. The parent7,8,9,l0-tetrahydro-6H- cyclohepta[b]quinoxaline thus obtained is thenoxidized by means of peracids, e.g. hydrogen peroxide in glacial aceticacid, peracetic acid, m-chlorperbenzoic acid, performic acid ormonoperphthalic acid, to the corresponding monoor dioxide depending uponthe molar proportion of oxidizing agent used.

The mono-, or dioxide, is then treated with a lower alkanoic acidanhydride to produce the corresponding 6- substituted-lower alkanoyloxyquinoxaline compound, or in the case of the dioxide the corresponding6,10-di(lower)alkanoyloxy quinoxaline compound, in a Bockelheiderearrangement. Peracid oxidiation of the lower alkanoyloxy compoundsaffords the desired lower alkanoyloxy substituted 7,8,9,10 tetrahydro 6Hcyclohepta[b]quinoxaline dioxides. Acid hydrolysis provides thecorresponding monoor di-hydroxy derivatives.

The rearrangement is conveniently conducted by heating the 7,8,9,10tetrahydro 6H cyclohepta[b]quinoxaline-S-oxide or ill-dioxide at anelevated temperature, e.g. from about 50 C. to the boiling point of thesolvent medium, with an excess of the desired lower alkanoyl anhydride.The excess anhydride generally serves as solvent medium. However,reaction-inert solvents, that is, solvents which do not react in anundesired manner with the reactants or products under the conditions ofthe reaction can be used. A minimum of laboratory experimentation willpermit the selection of suitable solvents for this reaction. Exemplaryof such solvents are dioxane, tetrahydrofuran, carbon tetrachloride,chloroform, methyl ether of diethylene glycol and the methyl ether ofethylene glycol. The reaction is run for a period of about 30 minutes,or longer if necessary, and the product recovered by removal of theexcess lower alkanoyl anhydride and/ or solvent medium.

Alternatively, the lower alkanoyloxy derivatives can be prepared asdescribed by Gordon, US. Pat. 2,921,937 which comprises reacting thehalogenated derivatives with a tertiary amine salt of the appropriatelower alkanoic acid. The amine salt is conveniently, but notnecessarily, formed in situ by simply mixing the amine and acid.Althrough any tertiary amine is suitable, pyridine, trimethylamine,triethylamine and dimethylaniline are favored because of theiravailability.

In general, an excess of the tertiary amine and lower alkanoic acid areused, about 2 to 3 moles of tertiary amine and about 4 to 5 moles oflower alkanoic acid per mole of dihalogenated compound, larger orsmaller ratios of amine and acid can be used but afford no advantages.Smaller ratios of amine and acid increase the length of time requiredfor the reaction. The liquid alkanes and halogenated alkanes serve assolvents for this reaction. Temperatures of from about to 120 C. andreaction periods of from about one to about 24 hours providesatisfactory yields.

The lower alkanoyloxy substituted 7,8,9,l0-tetrahydro-6H-cyclohepta[b]quinoxaline dioxides thus obtained serve asintermediates for the remaining novel products of this invention. Forexample, by acid hydrolysis the alkanoyloxy groups are converted tohydroxy groups. Suitable acid hydrolyzing agents are the mineral acids,e.g., hydrochloric, hydrobromic, sulfuric, nitric, phosphoric. Thereaction is generally conducted at a temperature of from about 10 C. toabout 50 C., and preferably at room temperature for periods of fromabout 1 to about 24 hours. The concentrated form of the acid is favoredsince it accelerates the rate of reaction and affords better yields thando lower concentrations.

The formyloxy derivatives are prepared by the acylation of thecorresponding monoor dihydroxy compound, e.g., by means of acetoformicacid, at a temperature of from about C. to about C., or by formylfluoride.

Acetoformic acid reagent suitable for the present process is prepared bymixing one volume of formic acid with two volumes of acetic anhydride.Approximately 72 mil. of this reagent is equivalent to one mole ofacetoformic acid anhydride. Excess of acetoformic acid reagent isemployed when higher reaction temperatures are employed since thereagent is decomposed in the presence of basic substances to provideacetic acid and carbon monoxide. This side reaction becomes increasinglyrapid above 10 C. Therefore, it is preferred to operate below thistemperature. As a practical matter, the range from 0 to 10 C. ispreferred for best results and economy. When operating in thistemperature range, from 1.5 to 2 milliliters of acetoformic acid reagentper gram of antibiotic reactant is satisfactory. An equimolecularproportion of the reagent is adequate at the lower level of thetemperature range.

Diluents which are non-reactive and do not catalyze decomposition of thereagent at the reaction temperature can sometimes be advantageouslyemployed in the present process. Illustrative of operable diluents arenon-hydroxyl containing solvents such as dioxane, toluene, benzene,dimethylformamide, ethyl acetate, methyl isobutyl ketone, acetone,pyridine, quinoline, etc. Hydroxylated solvents such as the loweralkanols and glycols are not satisfactory due to the tendency ofacetoformic acid reagent to react with these materials. It has beenfound that the present process is particularly adapted to the use ofpyridine as a solvent medium.

The hydroxy group (or groups) can be replaced by chloro and bromo byreaction with thionyl chloride, thionyl bromide, phosphorous tribromideor phosphorous 4 trichloride in the presence of a tertiary organic basesuch as pyridine, dimethyland diethylaniline.

The monohalo (chloro and bromo) derivatives of Formula 1 are preferablyprepared by halogenation of 7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline dioxide in a reaction-inertsolvent medium such as a liquid alkane, a liquid halogenated alkane andN-alkylated lower amide; e.g. n-hexane, chloroform, carbontetrachloride, N-methyl formamide, N,N-dimethylformamide. Molar ratiosof halogenating agent to 7,8,9,lO-tetrahydro-6H-cyclohepta[b]quinoxaline-5,ll-dioxide of 1:1 favor production of monohalogenatedderivatives. For the production of dihalogenated products molar ratiosof about 2 to 8 moles of halogenating agent to starting compound areused. Larger amounts of halogenating agent can be used but afford noapparent advantages. The reaction is conducted at a temperature of fromabout 15 to about C. for a preiod of from about 1 to about 3 hours andmost conveniently at the reflux temperature of the solvent medium forperiods of from about 1 to about 5 hours.

Suitable halogenating agents for the present process include bromine;chlorine; iodochloride; iodobromide; N- chloro, and N-bromo loweralkanoic acid amide, e.g. N- chlor, and N-bromacetamide; hydrocarbondicarboxylic acid imides, e.g. N-chloro-, N-bromosuccinimide,phthalimide, and the like and N-lower-alkanoyl anilines, e.g. N-bromoacetanilide, N-bromopropionanilide and the like; 3- chloro-,3-bromo-, 3,5-dichloro and 3,5-dibromo-5,5-dimethylhydantoin; pyridiniumperbromide and perchloride hydrohalides, e.g. pyridinium perbromidehydrobromide; pyridinium perchloride hydrochloride; and lower alkylhypochlorites, e.g. tertiary butylhypochlorite. It is obvious that, ingeneral, any halogenating agent employed in the art is operable, but theabove are preferred.

The products are thus obtained as their hydrohalide salts. The salts areconverted to the free bases by treatment with an alkaline reagent suchas an alkali metal or alkaline earth metal hydroxide, carbonate orbicarbonate.

The chloro and bromo groups are then converted by metathesis to cyano,mercapto, lower alkyl mercapto or lower alkoxy groups by a Williamsontype reaction using metallic salts of hydrogen cyanide, hydrogensulfide, lower alkyl mercaptans or lower alcohols as reactants. Thepreferred metal salts are those of the alkali metals, especially sodiumand potassium. Other metal salts such as the lead, zinc, magnesium saltscan also be used.

Alternatively, the methoxy and methyl-mercapto ethers are prepared bymethylation of the hydroxy or mercapto groups with dimethyl sulfate ordiazo methane according to known methods.

Oxidation of the hydroxy group or groups as the case may be produces thecorresponding monoor di-keto compounds (Formulae II and III). Suitableoxidizing agents are chromium trioxide, potassium dichromate andpotassium permanganate. The oxidation of the hydroxy group or groups canbe accomplished before, after or simultaneously with introduction of theN-oxide groups.

Dehydration of the monohydroxy compounds by suitable agents, e.g.,sulfuric acid, produces the corresponding monoand diunsaturatedcompounds (Formulae IV and V).

The novel carboxy and carbo(lower)alkoxy derivatives are prepared bycondensation of the appropriate 1,2-cycloheptanedione monoordicarbo(lower) alkoxy compound, e.g.,1,2-cycloheptanedione-3-ethylcarboxylate, 1,2-cycl0-heptanedione-3,7-diethylcarboxylate,w ith o-phenylenediamine, generallyin the presence of a small amount of acid such as concentratedhydrochloric acid, in a reaction-inert solvent followed by peracidoxidation to the monoor dioxide. Suitable solvents for this condensationare benzene, xylene, toluene and other aromatic hydrocarbons. Thereaction is conducted at an elevated temperature, preferably at thereflux temperature of the solvent system, with removal of water as byazeotropic distillation.

Acid hydrolysis of the carbo(lower)alkoxy derivatives with a mineralacid (hydrochloric, hydrobromic, sulfuric,

nitric, phosphoric) or with an alkali or alkaline earth hydroxide,bicarbonate or carbonate produces the carboxy derivative. Alternatively,the carboxy derivatives are produced by hydrolysis, preferably acidhydrolysis, of the corresponding cyano derivatives. Subsequentesterification of the acid derivatives provides the esters according toknown procedures.

The unsaturated compounds of Formulae IV and V participate inelectrophilic addition reactions with a variety of nucleophilic reagentssuch as the hydrogen halides, primary and secondary amines, hydrogencyanide, hypohalous acids, halogens and epoxide formation to producenovel compounds useful as antibacterial agents and as intermediates forthe production of a variety of products, many of which are antibacterialagents.

The acid addition salts are prepared by dissolving the free base in asuitable non-aqueous solvent, e.g., acetone, ether, lower aliphaticalcohols (ethanol, isopropanol) containing the desired acid, or to whichthe stoichiometric amount of the desired acid is added. Acid saltscontaining 1:2 and 2:1 molar ratios of acid to base are thus prepared.The alkali metal and alkaline earth metal salts of the monoand dicarboxysubstituted compounds of this invention are prepared by simpleneutralization of the acid derivative in aqueous solution with theappropriate metal hydroxide, carbonate or bicarbonate and recovered byprecipitation with a non-solvent, evaporation of the solvent or bylyophilization.

The novel products of this invention are valuable agents for the control(treatment and prophylaxis) of urinary tract and systemic infections inanimals including man and are of particular value against gram-negativeinfections both in vitro and in vivo. Further, these products areeffective in the control of air sacculitis (air sac disease) of poultry,and significantly promote growth and improve feed efficiency of domesticanimals, especially of poultry. In addition to the new compoundsdescribed herein the parent compound, 7,8,9,l-tetrahydro6H-cyclohepta[b]quinoxaline-3,ll-dioxide, has also been found effective for the samepurposes.

When used for such purposes compounds described herein can beadministered orally or parenterally, e.g. by subcutaneous orintramuscular injection, at a dosage of from about 1 mg./kg. to about100 mg./kg. of body weight. a

For parenteral administration dosages of from about mg./kg. to about 100mg./kg. of body weight are preferred. Vehicles suitable for parenteralinjection may be either aqueous such as water, isotonic saline, isotonicdextrose, Ringers solution, or non-aqueous such as fatty oils ofvegetable origin (cotton seed, peanut oil, corn, sesame),dimethylsulfoxide and other non-aqueous vehicles which will notinterfere with the therapeutic efiiiency of the preparation and arenon-toxic in the volume or proportion used (glycerol, propylene glycol,sorbitol). Additionally, compositions suitable for extempraneuspreparation of solutions prior to administration may advantageously bemade. Such compositions may include liquid diluents, for example,propylene glycol, diethyl carbonate, glycerol, sorbitol, etc.; bufferingagents, hyaluronidase, local anesthetics and inorganic salts to afforddesirable pharmacological properties. For oral administration dosages offrom about 1 mg./ kg. to about 60 mg./kg. of body weight are favored.This can be achieved by a number of methods including dosage unitformulations such as capsules, tablets, lozenges, troches, liquidmixtures and solutions. In the case of poultry and domestic animalsother methods include mixing with the feed, the preparation of feedconcentrates and supplements. Additionally, dilute solutions orsuspensions, e.g. a 0.1% solution, can be supplied for drinkingpurposes.

For prophylactic use, about 10 to about 100 mg./kg. of body weight dailyis administered. The above methods of administration are suitablealthough administration in the animals food, water or mineral mixture ismore convenient.

In still another modification of this invention the poultry are isolatedand subjected, in a confined space, to a fine dust of silica on which isdispersed or adsorbed one or more of the herein described compounds. Thesilica, comprising particles of up to 20 m or less size, is suspended inthe air breathed by the poultry.

The unique feed compositions of this invention are found to beparticularly valuable for use with poultry and especially for poultryinfected with chornic respiratory disease. A type of conventional feedmaterial which may be employed is recommended to contain roughly between50% and 80% of grains, between 0% and 10% animal protein, between 5% and30% vegetable protein, between 2% and 4% minerals together withsupplemental vitaminaceous sources. When a feed containing a majorproportion of these substances and a minor proportion of one of theherein mentioned drugs is employed, the poultry shows a markedimprovement, if not complete recovery, over the infection and reach thedesirable weight in a shorter period of time than usual with a markedlygreater feed efficiency. It should be noted that these valuable productseliminate, or at least minimize, the economic losses normally associatedwith chronic respiratory disease.

Further, the addition of a low level of one or more of the hereindescribed 7,8,9,l0-tetrahydro-6H-cyclo hepta[b]quinoxaline-5,ll-dioxidesand derivatives thereof to the diet of healthy animals, both ruminantand non-ruminant, such that these animals receive the product over anextended period of time, at a level of from about 1 mg./kg. to about 100mg./kg. of body weight per day, especially over a major portion of theiractive growth period, results in an acceleration of the rate of growthand improved feed efficiency. Included in these two classes of animalsare poultry (chickens, ducks, turkeys), cattle, sheep, dogs, cats,swine, rats, mice, horses, goats, mules, rabbits, mink, etc. Thebeneficial effects in growth rate and feed efficiency are over and abovewhat is normally ob- 0 tained with complete nutritious diets containingall the nutrients, vitamins, minerals and other factors known to berequired for the maximum healthy growth of such animals. The animalsthus attain market size sooner and on one less feed.

The herein described feed compositions have been found to beparticularly valuable and outstanding in the case of such animals aspoultry, rats, hogs, swine, lambs, cattle, and the like. In someinstances the degree of response may vary with respect to the sex of theanimals. The products may, of course, be administered in one componentof the feed or they may be blended uniformly throughout a mixed feed;alternatively as noted above, they may be administered in an equivalentamount via the animals water ratio. It should be noted that a variety offeed components may be of use in the nutritionally balanced feeds.

The resulting new feed compositions have marked effects on the rate ofgrowth and feed efliciency. Feed efficiency, an extremely importanteconomic factor in raising animals, may be defined as the number ofpounds of feed required to produce a pound gain in weight. The novelfeed supplements of this invention permit the use of higher energy,higher protein diets to obtain improved feed/ gain ratios and the use offeedstufis that at present are not utilized efliciently. Simply stated,the compositions of this invention when fed to animals are moreefliciently converted to animal body weight than prior art compositions.Any animal feed composition may be prepared to comprise the usualnutritional balance of energy, proteins, minerals, and vitamins togetherwith one or more of the quinoxaline-di-N-oxides described above. Some ofthe various components are commonly grains such as ground grain, andgrain by-products; animal protein substances, such as meat, and fishby-products; vitaminaceous mixtures, e.g. vitamin A and D mixtures,

7 ribofiavian supplements and other vitamin B complexes; and bone meal,limestone, and other inorganic compounds to provide minerals.

The relative proportions of the present compounds in feeds and feedconcentrates may vary somewhat, depending upon the compound, the feedwith which they are employed and the animal consuming the same. Thesesubstances are advantageously combined in such relative proportions withedible carriers to provide concentrates which may readily be blendedwith standard nutritionally balanced feeds or which may be usedthemselves as an adjunct to the normal feedings.

Dry pre-mixes containing these compounds are prepared containing from0.10 to about 10% of the active ingredient mixed with salt (sodiumchloride) and other minerals which it is desired to incorporate into thepoultry ration. This can then be fed on an ad libitum basis by adjustingthe proportion of active ingredient in the mixture to the average dailyconsumption per bird so as to provide the proper daily dose as specifiedabove. If prepared feed supplements are employed, the material can beadministered in admixture with the feed. Again a concentration range ofabout 0.10 to 10% of the drug in the feed is employed. However, higherproportions can be satisfactorily employed depending upon thepalatability of the product to the poultry. This can be readilydetermined by simple experimentation. It is sometimes convenient to mixthe daily dose with only a portion of the average daily allotment toinsure complete consumption of the dose. The balance of the daily feedsupplement can then be fed after consumption of the medicated portion inthe usual fashion. These methods are particularly useful forprophylactic treatment, but similar compositions can be employed fortherapeutic use.

In the preparation of concentrates a wide variety of carriers may beemployed containing the aforesaid drugs. Suitable carriers include thefollowing: soybean oil meal, corn gluten meal, cotton seed oil meal,sunflower seed meal, linseed oil meal, cornmeal, limestone and corncobmeal. The carrier facilitates uniform distribution of the activematerials in the finished feed with which the concentrate is blended.This is especially important because only a small proportion of thesepotent materials are required. The concentrate may be surface coated, ifdesired, with various proteinaceous materials or edible waxes, such aszein, gelatin, microcrystalline wax and the like to provide a protectivefilm which seals in the active ingredients. It will be appreciated thatthe proportions of the drug preparation in such concentrates are capableof wide variation since the amount of active materials in the finishedfeed may be adjusted by blending the appropriate proportion ofconcentrate with the feed to obtain the desired degree ofsupplementation. In the preparation of high potency concentrates, i.e.premixes, suitable for blending by feed manufacturers to producefinished feeds or concentrates of lower potency, the drug content mayrange from about 0.1 g. to 50 g. per pound of concentrate. Aparticularly useful concentrate is provided by blending 2 g. of drugwith 1 pound of limestone or 1 pound of limestone-soybean oil meal (1:1). Other dietary supplements, such as vitamins, minerals, etc. may beadded to the concentrates in the appropriate circumstances.

The high potency concentrates may be blended by the feed manufacturerwith proteinaceous carriers, such as soybean oil meal, to produceconcentrated supplements which are suitable for direct feeding toanimals. In such instances the animals are permitted to consume theusual diet of corn, barley and other fibrous grains and the like. Theproportion of the drug in these supplements may vary from about 0.1 to10 g. per pound of supplement.

The concentrates described may also be added to animal feeds to producea nutritionally balanced, finish feed containing from about 10 to about125 g. of the herein described compounds per ton of finished feed. Inthe case of ruminants, the finished feed should contain protein,

fat, fiber, carbohydrate, vitamins and minerals, each in an amountsufiicient to meet the nutritional requirements of the animal for whichthe feed is intended. Most of these substances are present in naturallyoccurring feed materials, such as alfalfa hay or meal, cracked corn,whole oats, soybean oil meal, corn silage, ground corn cobs, wheat bran,and dried molasses. Bone meal, limestone, iodized salt and traceminerals are frequently added to supply the necessary minerals, and ureato provide additional nitrogen.

As is Well known to those skilled in the art, the types of diets areextremely variable depending upon the purpose, type of feedingoperation, species, etc. Specific diets for various purposes are listedby Morrison in the Appendix of Feeds and Feeding, The MorrisonPublishing Company, Clinton, Iowa, 1959.

In the case of non-ruminant animals, such as hogs, a suitable feed maycontain from about 50 to of grains, 3 to 10% animal protein, 5 to 30%vegetable protein, 2 to 4% of minerals, together with supplementaryvitaminaceous sources.

The parent compound of the novel compounds of Formula 1 wherein R and Rare hydrogen, that is, 7,8,9,l0-tetrahydro-6H-cyclohepta[b]quinoxaline-5,11 dioxide, is devoid ofantibacterial activity. It is, therefore, unexpected to find that itsderivatives are surprisingly effective as antibacterials.

When used for the purposes described herein the quinoxaline-di-N-oxidescan, of course, be used in combination with other known drugs such asthe tetracycline-type antibiotics, carbomycin, neomycin, bacitracin andtylosin.

In addition to the above mentioned utilities, thequinoxaline-di-N-oxides described herein have unexpectedly been found toexhibit activity against antibiotic-resistant strains of microorganisms,such as antibiotic-resistant strains of Escherichia c011 originallyisolated from poultry and from man. Further, this unexpected activity issurprisingly applicable to microorganisms in which resistance toantibiotics has been induced, as by exposure to other microorganismsalready resistant to antibiotics.

The following examples are provided to illustrate in greater detail themanner of practicing the present invention. They are, however, not to beconsidered as limiting the scope thereof in any way.

EXAMPLE I 7, 8 ,9, l 0-tetrahydro-6H-cyclohepta [b] quinoxaline Asolution of o-phenylenediamine (108.0 g.) and cycloheptanedione (126.0g.) in benzene (2 liters) and concentrated hydrochloric acid (1 ml.) isrefluxed for 10 hours during which time the by-product, water (27 ml.),is collected in a Dean-Stark trap. The solution (black) is evaporated toabout one-third volume and chromatographed on a column of activatedalumina (Grade 1). Elution with benzene and removal of the benzene invacuo yields the crude crystalline product (147 g.).

Recrystallization from acetone/hexane yields the pure product, M.P.79.5-81.5 C.

Analysis.Calc. for C H N (percent): C, 78.75; H, 7.12; N, 14.13. Found(percent): C, 78.83; H, 6.99; N, 13.87.

U.V. (CH OH) x238 (e=35,000), x242(shoulder) (e=30,000), A307(shoulder)(e=7,940), x317 (6: 10,000), A326(shoulder) (6:7,730).

EXAMPLE II 7,8,9, 1 O-tetrahydro-6H-cyclohepta [b] quinoxaline-S-oxide Asolution of 7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline (198 g.)and m-chloroperbenzoic acid (200 g., in chloroform (2 liters) is allowedto stand at room temperature for 48 hours. The reaction mixture is thenwashed with a saturated solution of sodium bicarbonate, dried overanhydrous sodium sulfate, treated with activated charcoal, filteredthrough super-cel and evaporated to dryness. Crystallization of theresidue from acetone/hexane provides the product; M.P. 67-80 C. (129g.). Recrystallization from acetone/ hexane yields the pure product;M.P. 85.5 87 C.

Analysis.Calc. for C H ON (percent): C, 72.87; H, 6.59; N, 13.08. Found(percent): C, 72.83; H, 6.70; N, 13.11.

U.V. (CH OH) A244 (e=46,250), A325 (5:7,950), A340 (e=7,050).

EXAMPLE III 7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-5,11-dioxide A solution of 7,8,9,10 -tetrahydro-6H-cyclohepta[b]quinoxaline (214 g.) and m-chloroperbenzoic acid (500 g.) in chloroform(2 liters) is allowed to stand at room temperature for 3 days. Theby-produot, m-chlorobenzoic acid, is filtered ofl and the filtraterefluxed for 30 minutes then Washed repeatedly with a saturated solutionof sodium bicarbonate. Removal of the chloroform in vacuo gives thecrude product, a residue (347 g.) which is chromatographed on activatedalumina (Grade 1) using benzene as eluant. Removal of the benzene andcrystallization of the residue from acetone gives the product as yellowneedles; M.P. 160164 C. (dec.) (67 g.). Recrystallization from acetoneprovides the pure dioxide; M.P. 171-173 C.

Analysis.Calc. for C H O N (percent): C, 67.81; H, 6.13; N, 12.17. Found(percent): C, 67.97; H, 5.95; N, 11.95.

U.V. (H O) A239 (s=22,600), A267 (s=44,200), A348 14,300), A362(e=14,000).

EXAMPLE IV 6-acetoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline7,8,9,10-tetrahydro 6H cyclohepta[b]quinoxaline-S- oxide (5 g.) isdissolved in acetic anhydride (25 ml.) and refluxed for 2 hours using aSunbeam lamp as the source of heat. The excess acetic anhydride isremoved in vacuo and the residual gum chromatographed on a column ofacid-washed Florisil (synthetic magnesium silicate) using benzene aseluant. The product is collected and crystallized from acetone/hexane;M.P. 112116 C. (3.2 g.). Recrystallization from acetone/ hexane givesthe pure compound; M.P. 116.5117.5 C.

Analysis.Calc. for C H O N (percent): C, 70.29; H, 6.29; N, 10.93. Found(percent): C, 70.36; H, 6.30; N, 10.90.

U.V. (CH OH) A238 (e=32,000), shoulder A308 (e=6,220), A317 (e=7,700),shoulder A325 (e=6,030).

LR. (CHCl Principal absorption maxima 5.76, 7.33, 6.00, 840p.

The following lower alkanoyloxy derivatives are similarly prepared usingthe appropriate acid anhydride in place of acetic anhydride:propionyloxy, butyryloxy, isovaleryloxy, caproyloxy.

EXAMPLE V 6-acctoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-S,1 l-dioxide A solution of6-acetoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline (Example IV,50 g.) and m-chloroperbenzoic acid (100 g.) in chloroform (2 liters) isallowed to stand for 3 days at room temperature. The byproduct,m-chlorobenzoic acid, is removed by filtration and the filtrate thenrefluxed for 30 minutes. The solution is cooled and then washer,extracted repeatedly with saturated sodium bicarbonate solution anddried. Removal of the chloroform in vacuo gives a residue (65 g.) whichcrystallizes from acetone/hexane: M.P. 149-153 C. (dec.); 34 g.

An alysis.-Calc. for C H O N (percent); C, 62.49; H, 5.59; N, 9.75.Found (percent): C, 62.46; H, 5.81; N, 9.57.

LR. (CHCl Principal absorption maxima 5.71, 7.36, 7.56 4.

U.V. (CH OH) A235 (e=24,000), A266 34,000), A378 (e=11,33.0).

Oxidation of the remaining products of Example IV in the same mannerproduces the corresponding 5,11-dioxides.

EXAMPLE VI 6-hydroxy-7,8,9,l0-tetrahydro-6H-cyclohepta [b]quinoxaline-5,1 l-dioxide 6 acetoxy 7,8,9,10 tetrahydro 6H cyclohepta[b]quinoxaline-5,11-dioxide (20 g., Example V) is dissolved in concentratedhydrochloric acid (100 ml.) at room temperature. After 2 hours at roomtemperature the mixture is evaporated to dryness in vacuo. The residueis crystallized from acetone/hexane: (14.4 g.) M.P. 181184 C.

Recrystallization from chloroform/ hexane yields the pure product: M.P.187190 C.

AnaIysis.-Ca1cd. for C H O N (percent): C, 63.40; H, 5.73; N, 11.38.Found (percent): C, 63.41; H, 5.86; N, 11.29.

U.V. (CH OH) A234 (e=24,200), A267 (e=35,700), A374 (e=12,700).

By means of this procedure the remaining products of Example V arehydrolyzed to the same compound.

EXAMPLE VII6,10-diacetoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline7,8,9,10-tetrahydro 6H cyclohepta[b]quinoxaline-5, ll-dioxide (20 g.) isdissolved in acetic anhydride (100 ml.) and the solution refluxed for 45minutes. The solution is cooled, poured on to cracked ice and extractedwith chloroform. The chlorofrom solution is treated with activatedcharcoal, dried over anhydrous sodium sulfate and filtered. Removal ofthe chloroform in vacuo yields a dark gum (24.75 g.). The gum is takenup in benzene and chromatographed on a column of acid-washed Florisilusing benzene as eluant. Evaporation of the eluate gives the crudeproduct (9.65 g.) 'which is purified by crystallization fromacetone/hexane as prisms: M.P. 171- 173 C.

Analysis.Calc. for C1'7H18O4N2 (percent): C, 64.95; H, 5.77; N, 8.91.Found (percent): C, 64.91; H, 5.76; N, 8.97.

IR. (CHCl Principal absorption maxima 5.73, 7.30, 8.05-8.4, 9.16

U.V. (CH OH) A236 (e=32,700), A316 (6=7,100).

Substitution of acetic anhydride by the following anhydrides producesthe corresponding di(lower)alkanoyloxy compounds: anhydrides ofpropionic, butyric, valeric, isobutyric, caproic acid.

EXAMPLE VIII 6,10-diacetoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b]quinoxaline-5,1 l-dioxide 6,10 diacetoxy7,8,9,10-tetrahydro-6H-cyclohepta[b] qu1noxalme (50 g.), the titleproduct of Example VII, is dissolved in chloroform (1 liter),m-chloroperbenzoic acid (65 g., added and the mixture refluxed for IX.(CHCI Principal absorption maxima 5.75, 7.34, 7.52, 8.05-8.35 t.

U.V. (CH OH) x237 (e=23,800) x268 (e=22,500) A383 (e=8,400).

In like manner the remaining di(lower)alkanoyloxy compounds of ExampleVII are oxidized to their corresponding dioxides.

EXAMPLE IX 6,10-dihydroxy7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline-5, 1 1-dioxide A solution of 6,10 diacetoxy 7,8,9,1Otetrahydro- 6H cyclohepta[b]quinoxaline 5,11 dioxide (11.0 g.) (ExampleVIII) in methanol (300 ml.) and 20% sulfuric acid (100 ml.) is allowedto stand at room temperature for 12 days, during which time the productseparate as crystals (5.7 g.). The product is filtered off, washed withwater and recrystallized from acetone to provide the pure product; M.P.177178 C. (dec.).

Analysis.Calc. for C H O N (percent): C, 59.53; H, 5.38; N, 10.68. Found(percent): C, 59.80; H, 5.39; N, 11.07.

U.V. (CH OH) x236 (e=23,200) x268 (e=28,600) shoulder x360 (e=l0,480)x380 (e=11,500).

Similarly, acid hydrolysis of the remaining di(lower) alkanoyloxycompounds of Example VIII produces the same product.

EXAMPLE X 6,10-dicarbethoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b]quinoxaline To 1,2 cycloheptanedione 3,7 diethylcarboxylate (9.0 g.)dissolved in benzene (250 ml.), o-phenylenediamine (3.75 g.) and onedrop of concentrated hydrochloric acid are added and the mixturerefluxed for 3 hours, the by-product water being removed by means of aDean-Stark trap. The benzene is removed in vacuo and the residueextracted with chloroform. The crude product remaining after removal ofthe chloroform is chromatographed on a Florisil column using chloroformas eluant. The product is obtained as yellow crystals from acetone.

In like manner the following 6,10 di(carbo(lower) alkoxy) derivativesare prepared from the appropriate diester of 1,2 cycloheptanedione 3,7dicarboxylic acid; the dicarbornethoxy-, dicarbopropoxy-,dicarboisopropoxy-, dicarbovaleroxyand dicarbobutoxy-.

The products are converted to their respective dioxide by the procedureof Example III.

EXAMPLE XI 6,10-dicarboxy-7,8,9,10-tetrahydro-GH-cyclohepta[b]quinoxaline-5,1 l-dioxide 6,10 dicarbethoxy 7,8,9,10 tetrahydro 6Hcyclohepta[b]quinoxaline 5,11 dioxide g.) is dissolved in ethanol (250ml.) and 2 N HCl (50 ml.). The solution is refluxed for 6 hours thenconcentrated to /3 volume. The crystalline product separates on cooling.

The remaining products of Example X are hydrolyzed to the same productby this procedure.

Neutralization of the dicarboxylic acids with 1 and 2 molar proportionsof sodium, potassium, calcium and magnesium hydroxide provides thecorresponding metal salts.

EXAMPLE XII 6-bromo-7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-5,1l-dioxide To a solution of 7,8,9,10 tetrahydro 6H cyclohepta[b]quinoxaline 5,11 dioxide (23.0 g., 0.1 mole) in chloroform (250ml.) there is added, with stirring at room temperature, a solution ofbromine (16 g.) in chloroform (250 ml.) dropwise during 3 hours. Thebromine is added at such a rate that the bromine color disappears aftereach addition. Stirring is continued for 1 additional hour followingcompletion of addition. The

12 chloroform solution is washed with saturated sodium bicarbonatesolution and the product isolated by concentration of the chloroformsolution and crystallization from chloroform/ hexane.

Repetition of this procedure but substituting the proper halogen and theappropriate pentamethylene quinoxaline dioxide for the above reactantsyields the following compounds:

EXAMPLE XIII 6,10-dibromo-7,8,9,10-tetrahydro-6H-cyclohepta [b]quinoxaline-5, 1 l-dioxide To a solution of 7,8,9,10 tetrahydro 6Hcyclohepta [b]quinoxaline 5,11 dioxide (230.3 g., 1 mole) in 1500 ml. ofchloroform there is added 2.5 moles of bromine in 800 ml. of chloroform.The solution is refluxed for 2 hours during which time hydrogen bromideis evolved. The mixture is concentrated to half volume in vacuo thencooled to 1015 C. and filtered to remove the precipitated hydrogenbromide salt. The free base is obtained by slurrying the salt in anexcess of 5% aqueous sodium bicarbonate solution. The base is thenfiltered off, washed free of inorganic salts and dried.

By means of this procedure 6,10 dichloro 7,8,9,10- tetrahydro 6Hcyclohepta[b]quinoxaline 5,11 dioxide is prepared using chlorine inplace of bromine.

EXAMPLE XIV 6,10-diacetoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b]quinoxaline-5, 1 l-dioxide Method ATo a solution of 6.10 dibromo7,8,9,10- tetrahydro 6H cyclohepta[b]quinoxaline 5,11 dioxide (194 g.,0.5 mole) in 1500 ml. of chloroform there is added acetic acid (2.75mole) together with triethylamine (1.25 moles). The reaction mixture isrefluxed in the dark for 4 hours then concentrated to about 150 ml. toobtain a mixture of the epimeric 6,10-diacetoxy-7,8, 9,10 tetrahydro 6Hcyclohepta[b]quinoxaline-5,11- dioxides. The product is identical tothat of Method B.

Repetition of this procedure but substituting acetic acid by propionicacid, butyric and valeric acid produces the corresponding loweralkanoyloxy compounds.

Method B-A solution of 7,8,9,10 tetrahydro-6H-cyclohepta[b]quinoxaline-5,11-dioxide (23.0 g.) in acetic anhydride ml.)is refluxed for 45 minutes and then the excess acetic anhydride removedin vacuo to yield a dark residue. The dark color is removed by passing asolution of the residue in benzene; chloroform (1:1) through a column ofacid-washed Florisil. Evaporation of the effiuent gives a mixture of thestereoisomeric diacetates as a clear gum.

6,10 diacetoxy 7,8,9,l0 tetrahydro 6H cyclohepta[b]quinoxalinestereoisomeric mixture (12.0 g.) is dissolved in acetic acid (50 ml.)and peracetic acid (40%, 25 ml.) added dropwise with stirring during 20minutes.

After standing 7 days at room temperature the reaction mixture is pouredinto one liter of water and the mixture then extracted with chloroform(4x 250 ml.). The combined chloroform extract is washed repeatedly withsaturated sodium bicarbonate, dried with anhydrous sodium sulfate andthe chloroform removed in vacuo to yield a gum. Chromatography on anacid-washed Florisil (an activated magnesium silicate) column usingbenzene/chloroform mixtures yields the product which crystallizes fromacetone/hexane as yellow crystals.

Repetition of this method but using the appropriate acid anhydride inplace of acetic anhydride produces the followingl,3-di(lower)alkanoyloxy derivatives: dipropionyloxy-, dibutryloxy-,divaleryloxy, diisova1eryl-, oxyand dicaproyloxy.

EXAMPLE XV 6,10-dihydroxy-7,8,9,10-tetrahyclro-6H-cyclohepta[b]quinoXaline-5,l l-dioxide The title product of Example XIV,6,10-diacetoxy- 7,8,9,l-tetrahydro 6H cyclohepta[b]quinoxaline-5,lld-ioxide g.), is dissolved in methanol (300 ml.) and sulfuric acid (100ml.) added. The mixture is held at room temperature for 12 days, duringwhich time the product separates as crystals. The product is separatedby filtration, washed with water and recrystallized from acetone to givethe pure product.

The remaining products of Example XIV are similarly hydrolyzed to thesame dihydroxy compound.

Formylation of the dihydroxy compound following the procedure ofNeameyanov, et al., Ber. 67, 370 (1934) produces the diformyloxycompound. The same compound is obtained by adding 50 ml. of acetoformicacid reagent to 10 g. of the dihydroxy compound in 40 ml. of pyridine at0 C. After /2 hour in an ice bath the reaction mixture is poured intoice water and the product extracted with chloroform and recoveredtherefrom, after drying, by concentration to small volume.

EXAMPLE XVI 6-cyano-7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-S,1 l-dioxide 6 bromo 7,8,9,1O tetrahydro 6H-cyclohepta[b]quinoxaline-5,ll-dioxide (12.5 g.) is dissolved in a solution ofpotassium cyanide (3 g.) and K1 (0.5 g.) in methanol (1 liter) bystirring at room temperature. After standing 2 days at room temperaturethe solution is diluted with 2 liters of water and extracted withchloroform. Removal of the chloroform yields the product as a solid.

The following cyano derivatives of Formula I wherein R is CN aresimilarly prepared from the appropriate bromo or chloro derivatives ofExamples XII and XIII. In the case of the dicyano derivative theproportion of potassium cyanide used is doubled.

6-carboxy-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-S ,1l-dioxide 6-cyano-7,8,9,10-tetrahydro 6Hcyclohepta[b]quinoxaline-5,ll-dioxide (5.5 g.) is dissolved in asolution of acetic acid (100 ml.) and 10% hydrochloric acid (50 ml.) andrefluxed for 2 hours. Removal of the solvent affords the solid product.

The cyano derivatives of Example XVI are likewise hydrolyzed to theirrespective acids. The compounds bearing lower alkanoyloxy or carbo(lower)alkoxy groups are, of course, hydrolyzed to the precursor hydroxyand carboxy derivatives. They are reacylated or re-esterified accordingto known procedures.

The carboxy compounds thus produced are converted to their sodium,potassium, calcium and magnesium salts by neutralization in aqueoussolution with the appropriate metal hydroxide. The salts are recoveredby lyophilization.

EXAMPLE XVIII 6-carbomethoxy-7,8,9,l0-tetrahydro-6H-cyclohepta[b]quinoxaline-5,l l-dioxide 6 carboxy 7,8,9,l0 tetrahydro 6H cyclohepta[b]quinoxaline-5,ll-dioxide (5.5 g.) is dissolved in a mixture ofchloroform (200 ml.), methanol (5 ml.) and concentrated sulfuric acid (2ml.) and refluxed for 16 hours. The solution is washed with water (3x500 ml.), dried over anhydrous sodium sulfate and evaporated to a gum.Crystallization from acetone/hexane gives the pure product.

In like manner the carboxy acid derivatives of Example XVII areconverted to their corresponding methyl esters. Substitution of methanolby ethanol, propanol, n-butanol produces the corresponding lower alkylesters.

EXAMPLE XIX 6,l0-dicarbethoxy-7,8,9, lO-tetrahydro-6H-cyclohepta [b]quinoxaline To 1,2 cycloheptanedione 3,7 diethylcarboxylate (9.1 g.)dissolved in benzene (250 ml.), o-phenylene-diamine (3.75 g.) and onedrop of concentrated hydrochloric acid are added and the reactionmixture refluxed for 3 hours, the by-product water being removed bymeans of a Dean-Stark trap. The benzene is removed in vacuo and theresidue extracted with chloroform. The crude product remaining afterremoval of the chloroform is chromatographed on a column of acid-washedFlorisil using chloroform as eluant. The product is obtained as yellowneedles from acetone. Further crystallization from acetone yields thepure product.

The following 6,10 di(carbo(lower) alkoxy) derivatives are similarlyprepared from the appropriate 1,2-cycloheptanedione3,7-di(lower)alkyl-carboxylate; dicarbomethoxy-, dicarbobutoxy-,dicarboisopropoxy-, dicarbocaproyloxy-.

EXAMPLE XX 6, 1 0-dicarbethoxy-7,8,9, l O-tetrahydro-6H-cyclohepta [b]quinoxaline-S-oxide A solution of 6,10 dicarbethoxy-7,8,9,lO-tetrahydro-6H-cyclohepta[b]quinoxaline (Example XIX, 8.2 g.) in chloroform (100ml.) is added dropwise during 30 minutes to a solution ofm-chloroperbenzoic acid (5.5 g., in chloroform (50 ml.) with stirring.The reaction mixture is held overnight at room temperature then washedseveral times with saturated sodium bicarbonate solution and dried withanhydrous sodium sulfate. The crude product obtained by removal of thechloroform in vacuo is chromatographed on a column of acid-washedFlorisil. Elution with chloroform yields the product which crystallizesfrom acetone/hexane. Further recrystallization produces the purecompound.

Substitution of the remaining esters of Example XIX for the dicarbethoxyester in the above process produces the corresponding 5-oxides.

EXAMPLE XXI 6,10-dicarbethoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline-S, l l-dioxide 6,10-dicarbethoxy 7,8,9,10 tetrahydro 6Hcyclohepta[b]quinoxaline (Example XIX, 22.2 g.) is dissolved inchloroform (250 ml.). m-Chloroperbenzoic acid (50.0

g.) is then added to the solution and the mixture allowed to stand forthree days at room temperature. The by-product, m-chlorobenzoic acid, isfiltered off and the filtrate washed with a saturated sodium bicarbonatesolution. The residue remaining after evaporation of the solvent iscrystallized from acetone/hexane.

1.R. (CHCl Principal absorption maxima 5.75, 8.0 and 8.75

The remaining esters of Example XIX are likewise converted to theircorresponding dioxides.

EXAMPLE XXII 6-bromo-7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-S,1 l-dioxide 6 hydroxy 7,8,9,10 tetrahydro 6H cyclohepta[b]quinoxaline-5,l1-dioxide (12.4 g.) dissolved in a mixture of pyridine(50 ml.) and chloroform (100 ml.) is treated with phosphorus tribromide(15 g.). The mixture is allowed to stand at room temperature for 24hours then Washed with water (3X 500 ml.). The product is isolated fromthe residue obtained after removal of the chloroform by vacuumdistillation.

Following this procedure the dihydroxy compound of Example X isconverted to 6,10-dibromo-7,8,9,IO-tetrahydro-6H-cyclohepta [b]quinoxaline-S, l l-dioxide.

EXAMPLE XXIII 6-me'thy1merca-pt0-7,8,9,10tetrahydro6H-cyclohepta[b]quinoxaline-5,1 l-dioxide 6 bromo 7.8.9.10 tetrahydro 6H-cyclohepta[b]quinoxaline-5,11-dioxide (25 g.) is added to a solution of sodiummethylmercaptide (10 g.) in ethanol (1 liter). The mixture is stirredfor 48 hours at room temperature then poured into water (3.1) and theresulting solution extracted with chloroform. The chloroform solution isdried over anhydrous sodium sulfate and evaporated to a gum. Theresidual gum is crystallized from acetone/hexane to yield thecrystalline product.

This procedure is repeated but using the appropriate mercaptide and thehalogenated pentamethylene quinoxaline dioxides of Examples XII and XIIIto produce the following compounds:

EXAMPLE XXIV Lower alkoxy substituted pentamethylene quinoxalinedioxides Repetition of the procedure of Example XXIII but using theappropriate sodium alkoxide as reactant in place 16 of sodium methylmercaptide produces the following compounds:

EXAMPLE XXV 7,8-dihydro-6I-I-cyclohepta [b] quinoxaline- 5,11-dioxidewith anhydrous sodium sulfate then evaporated in vacuo.

The residue, crystallized from chloroform:hexane, gives the product asbright yellow crystals; M.P. 158161 C. Recrystallization fromchl0rOf0rm:hexane alfords the pure product; M.P. 162-164 C.

Analysis.-Calc. for C H O N (percent): C, 68.41; H, 5.30; N, 12.27.Found (percent): C, 68.43; H, 5.30; N, 12.24.

U.V. (CH OH) x242 (E=15,600) A288 (e=34,400) x380 (=1l,400).

Application of this procedure to the hydroxy substituted compounds ofExamples XII, XVI, XVII, XVIII, XXIII and XXIV produces thecorresponding unsaturated compounds having the formula:

EXAMPLE XXVI 6-bromo-10-carbethoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline-5,1 l-dioxide To6-carbethoxy-7,8,9,IO-tetrahydro 6H cyclohepta[b]quinoxaline-5,11-dioxide (15.0 g.) in chloroform (250 ml.) at refluxis added a solution of bromine (8 g.) in chloroform ml.) dropwise during30 minutes. After an additional 30 minutes at reflux the solution iscooled, washed with saturated sodium bicarbonate and dried withanhydrous sodium sulfate. Evaporation of the chloroform in vacuo at30-40 C. provides the product.

In like manner the methyl, isopropyl, n-butyl and amyl esters areprepared from the appropriate 6-carb0(lower)- alkoxy-7,8,9,IO-tetrahydro6H cyclohepta[b]quinoxaline-5,11-dioxide. Acid hydrolysis of thecarbo(lower alkoxy groups by the procedure of Example XI produces thecorresponding acid.

I 7 EXAMPLE XXVII 6-carboxy-7,8-dihydro-6H-cyclohepta[b] quinoxaline-S,1 l-dioxide 6acetoxy-7,8-dihydro-6H-cyclohepta[b] quinoxaline-S ,1l-dioxide 6-bromo 7,8 dihydro-6H-cyclohepta[b]quinoxaline- 5,11-dioxide(Example XXV, 4.7 g.) is treated with acetic acid and triethylarnineaccording to Method A of Example XIV to produce the title compound.

Hydrolysis of the acetoxy group according to the procedure of Example VIatfords the corresponding 6-hydroxy compound.

EXAMPLE XXIX 6-carbethoxy-7 8,9, 1 O-tetrahydro- 6H-cyclohepta [b]quinoxaline-S, l l-dioxide One drop of concentrated hydrochloric acid isadded to a solution of 1,2-cycloheptanedion-3-ethylcarboxylate (9.8 g.and o -phenylenediamine (3.75 g.) in benzene (250 ml.). The solution isrefluxed for three hours, the byproduct water being removed by means ofa Dean-Stark trap. The benzene is evaporated in vacuo and the residuetaken up in chloroform. The residue remaining after removal of thechloroform is chromatographed on a column of acidwashed Florisil usingchloroform as eluant. The product is obtained by evaporation of theeluate.

Oxidation of the product by the procedure of Example VIII provides thetitle product.

Replacement of 1,2-cycloheptanedione 3 ethylcarboxyla-te by thehomologous methyl-, propyl-, and caproyl esters produces thecorresponding 6-carbo(lower)alkoxyquinoxaline-S, 1 l-dioxides.

EXAMPLE XXX 6-acetoxy-10-carbethoxy-7, 8 ,9 l -tetrahydro-6H-cyclohepta[b] quinoxaline-S, l l-dioxide Application of the procedure of Method Aof Example XIV to 6 bromo-10-carb,ethoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinoxaline-5,1l-dioxide provides the title compound.

Acid hydrolysis of the product according to the procedure of Example VIaffords 6-hydroxy-10-carboxy-7,8,9,10-

tetrahydro 6H cyclohepta[b]quinoxaline-5,1l-dioxide.

Dehydration by the procedure of Example XXV affords6-carboxy-7,8-dihydro 6H cyclohepta[b]quinoxaline- 5,1 l-dioxide.

EXAMPLE XXXI 6-oxo-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-S,1 l-dioxide 6-hydroxy-7,8,9,'1o tetrahydro 6H cyclohepta[b]quinoxaline-5,11-dioxide (6.9 g.) is dissolved in alcoholfree chloroform(200 ml.) and shaken with a solution of chromium trioxide (10 g.) inwater (50 ml.) for 12 hours. The chloroform layer is separated, washedwith water, dried with anhydrous sodium sulfate and evaporated in vacuoto give the desired product.

Similarly, the hydroxy derivatives of Examples XII, XVI, XVII, XVIII,XXIII and XXIV are oxidized to the corresponding oxo-derivatives.

18 EXAMPLE XXXII 6-oxo-10-acetoxy-7,8,9,l0-tethydro-6I-I-cyclohepta[b]quinoxaline-5,1 l-dioxide 6-oxo-l0-bromo-7,8,9,10-tetrahydro 6Hcyclohept'a [b] quinoxaline-5,1l-dioxide (3.2 g., 0.01 mole) is treatedwith acetic acid (0.05 mole) and triethylarnine (0.03 mole) according tothe procedure of Example XIV, Method A, to give the title product.

Repetition of the procedure of this example but using propionic andbutyric acids in place of acetic acid producesthe correspondingalkanoyloxy compounds.

EXAMPLE XXXIII 6-oxo-10-hydroxy-7,8,9, lO-tetrahydro-6H-cyclohepta- [b]quinoxaline-S ,1 l-dioxide Acid hydrolysis of the title compound ofExample XXXII according to the procedure of Example VI affords the titlecompound.

EXAMPLE XXXIV 6,10-dioxo-7,8,9,IO-tetrahydro-6H-cyclophepta[b]-quinOxaline-S ,1 l-dioxide To a solution of 6,10-dihydroxy7,8,9,10-tetrahydro- 6H-cyclohepta[b]quinoxaline-5,1l-dioxide (6.55 g.)in alcohol free chloroform (20 ml.) is added a solution of chromiumtrioxide (20 g.) in Water ml.). The mixture is shaken for 12 hours. Thechloroform layer is separated, washed with water and dried withanhydrous sodium sulfate. Evaporation of the solvent in vacuo gives theproduct.

EXAMPLE XXXV 8H-cyclohepta [b] quinoxaline-S ,1 l-dioxide 6,10 dihydroxy7,8,9,10 tetrahydro-6H-cyclohepta- [b]quinoxaline-5,1l-dioxide (3.0 g.)is added to concentrated sulfuric acid (25 ml.). A vigorous reactionoccurs and the dark green solution which forms is allowed to stand atroom temperature for 30 minutes. The solution is then poured ontocrushed ice (400 g.). When the ice is completely melted the solution isextracted with chloroform. The chloroform extract is washed with water,

dried over anhydrous sodium sulfate and the chloroform removed in vacuo,to give the product (1.2 g.).

\EXAMPLE XPQiVI Acid addition salts EXAMPLE XXXV II The eflicacy of theherein described cyclohepta[b]- quinoxaline-5,11-dioxides in promotingthe growth of chicks is demonstrated in a series of tests conducted withVantress-White-rock chicks. In these tests, the day-old chciks aredivided into 4 lots of 25 each of one sex, and kept in electricallyheated brooders on raised wire floors. each lot is assigned to adifferent compartment and fed a basal diet supplemented with 0.05% byweight of 6,10- dihydroxy 7,8,9,10 tetrahydro 6H cyclohepta[b]-quinoxaline-5,11-dio)dde. A control in which no supplementation is addedis also run. The growth and feed efliciency responses of each group areobserved to 8 weeks of age. The composition of the basal ration isindicated below. The supplements are added to the diet in premix form atthe expense of yellow corn meal.

Ingredient: Percent of mix Yellow corn meal 56.65 Soybean oil meal (50%protein) 33.75 Alfalfa meal (17% protein) -u 2.00 Stabilized animal fat3.50 Multi-phos (dicalcium phosphate) 2.00 Iodized salt 0.50 Tracemineral vitamin premix 0.60 Limestone 1.00

Total 100.00

1 Trace mineral vitamin premix.

Vitamin5% of feed: Percent of premix Vitamin A- 10 Vitamin D 3O00 10Choline chloride (25%) 35.25

Niacin (80%) 0.55 Calcium pantothenate (45%) 10 Riboflavin-4 0.39 B12600.2 Vigofac-6 30 Fine corn meal 3.75

Mineral mix Percent of 0.05 percent of feed: mineral premix Manganese24.0 Iodine 0.48

Iron 8.0

Copper 0.8 Zinc 0.04

Cobalt 0.08

Vigofac is the registered trademark of Chas. Pfizer & Co., Inc. forunidentified poultry and livestock growth factors obtainable fromStreptomyces fermentation sources.

The chicks are observed daily, weighed weekly and the average gain perbird, the growth index and feed efficiency determined.

The 6,10-dihydroxy-7,8,9,10-tetrahydro-6H-cyclohepta-[b]quinoxaline-5,11-dioxide supplemented diet is found to produce asignificant increase in weight gained and a substantial improvement infeed efficiency.

Repetition of this procedure but using the followingquinoxaline-5,1l-dioxides produces similar results:

and 0.025% by weight of feed. In each case improvement in growth andfeed efficiency is noted.

EXAMPLE XXXIX Groups of baby pigs are fed on a completely balanced dietpreviously proven highly useful for this type of animal. These animals,weaned at approximately six to eight days of age, are fed over severaldays on a prestarter diet containing 60.1% dry skimmed milk, 10.1%soybean oil, 9.9% glucose hydrate, 1.1% brewers yeast, 0.4% ofoxytetracycline feed supplement containing 5 g./lb. of oxytetracycline,16% soybean oil meal and a complete vitamin and mineral supplement. Thepigs are then fed a starter diet of fine ground yellow corn, rolledoats, soybean oil meal, glucose hydrate, dicalcium phosphate, iodizedsalts, soybean oil, oxytetracycline supplement, vitamin mix and mineralmix. The starter feed for some of the groups is supplemented with g./tonof 6-hydroxy- 7,8,9,10 tetrahydro 6H cyclohepta[-b]quinoxaline-S,ll-dioxide. This feed is given the animals when they are about threeweeks old and continued over a total of four weeks.

The supplemented diet is found to result in economically importantweight gain and significant improvement in feed efficiency.

EXAMPLE XXXX Five-week-old chickens are infected with coliform airsacculitis by injecting 1.0 ml. of a 24-hour broth culture containing 2different antibiotic (tetracyline) resistant, avian pathogenic strainsof Escherichia coli into the left posterior thoracic air sac. Thequinoxaline-di-N-oxide test compound is administered in the feed, themedicated ration being fed for a 5-day period starting 2 days prior tothe coliform injection. The feed is tendered to the birds for imbibitionad libitum. Twenty-five hundred grams of medicated feed containing 0.05%by weight of the test compound is provided per 10 birds. When this isall consumed, non-medicated basal feed is given to the end of the trial.

The chickens are checked as to mortality, weight change, feedconsumption preand post-injection and, after sacrifice 3 dayspost-injection for air sac lesions. The followingquinoxaline-di-N-oxides are tested:

7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-5,1 1-

dioxide 6-hydroxy-7,8,9, 10-tetrahydro-6H-cyclohepta [b]quinoxaline-5,11-dioxide 6,10-dihydroxy-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-5,1l-dioxide 6-oxo-10-hydroxy-7,8,9, 10-tetrahydro-6H-cyclohepta [b]quinoxaline-5,1 l-dioxide 6, l0-diacetoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-5,1 l-dioxide6,10-dimethoxy-7,8,9,10-tetrahydro-6H-cyclohepta [b]quinoxaline-5,11-dioxide 6-carbethoxy-7,8,9,10-tetrahydro-6H-cyclohepta[b] quinoxaline-S ,1 l-dioxide6-bromo-10-oxo-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-5,1l-dioxide 6-mercapt0-7,8,9, 10-tetrahydro-6H-cyclohepta [b]quinoxaline-S ,1 l-dioxide 6-carboxy-7,8,9, 10-tetrahydro-6H-cyclohepta[b] quinoxaline-5,11-dioxide sodium salt 6-chloro-7,8,9,10-tetrahydro-6H-cyclohepta [b] quinoxaline-5,11-dioxide hydrochloride6-hydroxy-7,8,9, 10-tetrahydro-6H-cyclohepta [b]quinoxaline-5,11-dioxide sulfate 6-carboxy-7,8-dihydro-6H-cyclohepta [b]quinoxaline- 5,11-dioxide sodium salt All compounds are effective inreducing mortality, lesion scars and increasing body weight and feedconsumption. Effective control of the infection is realized.

EXAMPLE XXXXI Five-week old chickens are inoculated into the leftposterior thoracic air sac with 0.5 ml. of a 24-hour broth culturecontaining 2 different antibiotic (tetracycline) resistant, avianpathogenic strains of E. coli. The test compound is administered bysubcutaneous injection of an aqueous solution into the upper cervicalregion at 21 the time of infection. Observations on mortality, feedconsumption post-injection, weight change, and, after sacrifice, air saclesion scores are made. Ten birds are used for each test.

Effective control of the coliform infection is observed particularly atdosage levels of approximately 10 mg./kg. body weight and higher withthe following 7,8,9,l-tetrahydro-6H-cyclohepta [b] quinoxaline-S, ll-dioxides.

Compound: Dose mg./kg. 6,10-dihydroxy 10 6,10-diacetoxy 1O6-l0-dibutyryloxy 10 6,10-dibromo 1 10 6,10-dibromo 6-hydroxy 56-hydroxy 2.5 6-oxo l0 6-acetoxy-3-carbethoxy l0 6-acetoxy-3-carbethoxy20 6-carb0Xy 2 1O 1 Used as hydrochloric salt.

2 Used as sodium salt. and 6-carboxy-7,8-dihydro-6H-cyclohepta [b]quinoxaline- 5,11-dioxide sodium salt.

EXAMPLE XXXXII The relative efficacy of the quinoxaline-di-N-oxidesagainst Mycoplasma gallisepticum infection in chicks is demonstrated byexposing siX-week-old chicks, divided into groups of birds to theMycoplasma infection by injection of 0.5 m1. of a 72-hour broth culture,diluted with an equal volume of sterile heart infusion broth into theleft posterior thoracic air sac. The test compound is administered inthe basal feed at a level of 0.05%. Medication is started 43 hoursbefore exposure and continued for 72 hours post exposure for a total of3 days medication. Two replicates of each test are run.

The Mycoplasma infection alone exerts a pronounced effect on the lesionscore. Significant improvement of this characteristic reaction isobtained with 6,10-dihydroxyand 6,10-diacetoxy 7,8,9,10tetrahydro-6H-cyclohepta [b]quinoxaline-5,1l-dioxide and with theremaining compounds of this invention.

Similar results are obtained when this procedure is repeated but usingN-type PPLO and Mycoplasma iners as the infecting organisms.

R1 0 ll y y u 0 R2 0 R2 0 and l i/ 0 0 IV V wherein R is selected fromthe group consisting of hydroxy, lower alkoxy, mercapto, lower alkylmercapto, cyano, carboxy, carbo (lower)alkoxy, lower alkanoyloxy, chloroand bromo, R is selected from the group consisting of hydrogen and R andthe non-toxic mineral acid 22 addition salts thereof and, when either orboth of R and R is carboxy, the alkali metal and alkaline earth metalsalts thereof.

2. The method of claim 1 wherein the compound is administered orally.

3. The method of claim 1 wherein the compound is administeredparenterally.

4. A method for promoting growth and improving feed efficiency ofanimals which comprises administering to said animals an effectivegrowth promoting amount of a compound selected from the group consistingof 7,89,10- tetrahydro 6H cyclohepta[b]quinoxaline 5,1l-dioxide; andthose having the formulae wherein R is selected from the groupconsisting of hydroxy, lower alkoxy, mercapto, lower alkyl mercapto,cyano, carboxy, carbo(lower) alkoxy, lower alkanoyloxy, chloro andbromo, R is selected from the group consisting of hydrogen and R and thenon-toxic mineral acid addition salts thereof and, when either or bothof R and R is carboxy, the alkali metal and alkaline earth metal saltsthereof.

5. An animal feed composition comprising a nutritionally balanced animalfeed containing a growth promoting amount of a compound selected fromthe group consisting of 7,8,9,10 tetrahydro-6H-cyclohepta[b1quinoxaliue-5,1l-dioxide and those having the formulae and 2 R2 2wherein R is selected from the group consisting of hydroxy, loweralkoxy, mercapto, lower alkyl mercapto, cyano, carboxy, carbo(lower)alkoxy, lower alkanoyloxy, chloro and bromo, R is selected fromthe group consisting of hydrogen and R and the non-toxic mineral acidaddition salts thereof and, when either or both of R and R is carboxy,the alkali metal and alkaline earth metal salts thereof.

6. The animal feed composition of claim 5 wherein the compound ispresent at a level of from about 10 g. to about g. per ton of feed.

References Cited UNITED STATES PATENTS 2,626,259 1/1953 Landquist et al260-250 ALBERT T. MYERS, Primary Examiner FREDERICK E. WADDELL,Assistant Examiner Ting"? UNITED STATES PATENT OFFICE CERTIFICATE OFCORRECTIQN Patent No. 356uO93 Dated February 97 James David JohnstonInventor(s) It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below001. 5, line 21, "1:2" should read 1:1

line 39, "3" should read 5 Col. 11, line 1, "1.x." should read LR. Col.13, line 14, "d11sovalery1-, oxy-" should read diisovaleryloxy Col. 1line 5 4, "100" Should read lOOO Col 16, line 19, the blank space underR should read -OCOC H C01. 18, i'i e 26, "20" should read 200 Col 21,line 36, "H3" should read '48 Col. 22, lined; 22-28,

" @:N should read Signed and sealed this 10th day of August 1971.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E. SCHUYLER, JR. Attesting OfficerCommissioner of Patents

